This project addresses the genetic control of normal palate development in mice and humans by concentrating on the molecular identification of a gene responsible for recessive, lethal, isolated cleft palate in the mouse. Genetic- and molecular-mapping studies in the PI's laboratory with radiation-induced p-locus deletions in the mouse have identified a locus (cpl) that is necessary for proper palate development in the late- gestation fetus. Animals deleted for this gene manifest a clefting of the secondary palate that is similar to isolated cleft-palate defects observed in humans. These mapping studies show a complete correlation between the cleft-palate defect and alterations at the Gabrb3 locus, which encodes the 133 subunit of the GABAA receptor, thus suggesting that Gabrb3 might be cpl. To test this hypothesis, the wild-type Gabrb3 gene will be introduced back into these mutants, by transgenic-mouse technology, to ascertain whether it can correct the developmental defect. In parallel with these studies, the physical map around the cpl locus will be completed in case the gene-transfer experiments indicate that another candidate gene (i.e., not Gabrb3) be identified by positional- cloning techniques. Mouse fetuses homozygously deleted for cpl fail to undergo the process of palate-shelf elevation and reorientation. The RNA expression profiles of both wild-type and mutant palates will be studied by RT-PCR and RNA in situ hybridization to-analyze the temporal and spatial expression of the genes that encode others of the fifteen known GABAA receptor subunits to identify a specific receptor subtype necessary for normal palate development in the mouse. In addition, in case the (l) phenotype rescue experiments require wild-type mouse Gabrb3 regulatory sequences to direct spatially- and temporally-appropriate transgene expression or, (2) further data indicate that Gabrb3 is not cpl, we will use wild-type mouse YACs and cosmids to clone the genomic interval that contains cpl. Because defects in facial development are among the most common birth defects and are often substantial phenotypic components of a wide variety of inherited or environmentally induced clinical syndromes, molecular isolation of a gene absolutely required for the initial phases of palate formation would be an important first step in the analysis of this process. Likewise, the determination of whether a neurotransmitter receptor is involved in these developmental interactions should lead to an intensification of future research on non-neuronal roles for these molecules during embryonic and fetal development, and would provide yet another class of molecules to study in the context of environmentally induced fetal malformations.